Methods of suppressing microglial activation

ABSTRACT

Methods of suppressing the activation of microglial cells in the Central Nervous System (CNS), methods of ameliorating or treating the neurological effects of cerebral ischemia or cerebral inflammation, and methods of combating specific diseases that affect the CNS by administering a compound that binds to microglial receptors and prevents or reduces microglial activation are described. Also described are methods of screening compounds for the ability to suppress or reduce microglial activation.

RELATED APPLICATION INFORMATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/260,430, filed Mar. 1, 1999, which in turn claims thebenefit of U.S. Provisional Application No. 60/077,551, filed 11 Mar.1998, the disclosures of both of which are incorporated by referenceherein in their entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under NIH grantsNS368087-01A2, K08NS01949, and RO3 AG16507-01. The Government hascertain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to method of suppressing the activation ofmicroglial cells in the Central Nervous System (CNS), methods ofreducing or suppressing the activation of glial or microglial cells,methods of ameliorating or treating the neurological effects of cerebralischemia or cerebral inflammation, methods of combating specificdiseases that affect the CNS by administering a compound that binds tomicroglial receptors and prevents or reduces microglial activation, andmethods of screening compounds for the ability to prevent or reducemicroglial activation.

BACKGROUND OF THE INVENTION

The Central Nervous System (CNS) has long been considered to be a siteof relative immune privilege. However, it is increasingly recognizedthat CNS tissue injury in acute and chronic neurological disease may bemediated by the CNS inflammatory response. The CNS inflammatory responseis primarily mediated by inflammatory cytokines.

Apolipoprotein E (ApoE) is a 299 amino acid lipid-carrying protein witha known sequence (Rall et al., J. Biol. Chem. 257:4174 (1982); McLean etal., J. Biol. Chem. 259:6498 (1984). The complete gene for human ApoEhas also been sequenced (Paik et al., Proc. Natl. Acad. Sci. USA 82:3445(1985). ApoE sequences from at least ten species have been determined,and show a high degree of conservations across species, except at theamino and carboxyl termini. Weisgraber, Advances in Protein Chemistry45:249 (1994).

Human ApoE is found in three major isoforms: ApoE2, ApoE3, and ApoE4;these isoforms differ by amino acid substitutions at positions 112 and158. The most common isoform is ApoE3, which contains cysteine atresidue 112 and arginine at residue 158; ApoE2 is the least commonisoform and contains cysteine at residues 112 and 158; ApoE4 containsarginine at residues 112 and 158. Additional rare sequence mutations ofhuman ApoE are known (see, e.g, Weisgraber, Advances in ProteinChemistry 45:249 (1994),at page 268-269). The presence of ApoE4 has beenassociated with risk of developing sporadic and late-onset Alzheimer'sdisease (Strittmatter et al., Proc. Natl. Acad. Sci. USA 90:1977-1980(1993)).

ApoE plays a role in cholesterol metabolism and has also been reportedto have immunomodulatory properties. It is secreted by macrophages afterperipheral nerve injury and by astrocytes and oligodendrocytes (glialcells) after Central Nervous System (CNS) injury.

SUMMARY OF THE INVENTION

The present invention is based on the finding that microglial activationcan be reduced or suppressed using peptides that comprise the receptorbinding site sequence of Apolipoprotein E. Thus, the present inventionprovides methods and compositions for treating CNS disease states inwhich glial or microglial activation occurs, and in which glial ormicroglial activation contributes to the deleterious signs and/orsymptoms associated with the specific disease state.

The present invention is further based upon the identification of theaforesaid receptor as a high affinity Apolipoprotein E receptor, withthe binding characteristics of the LRP/α2M receptor.

In view of the foregoing, a first aspect of the present invention is amethod of suppressing glial or microglial activation in a mammal byadministering a compound that binds to glial or microglial cells at theLRP/α2M receptor (the receptor bound by a peptide of SEQ ID NO:3 or SEQID NO:6). The compound is administered in an amount that reduces glialor microglial activation compared to activation that which would occurin the absence of the compound.

A further aspect of the present invention is a method of amelioratingsymptoms associated with CNS inflammation by administering a compoundthat binds to glial or microglial cells at the LRP/α2M receptor (thereceptor bound by a peptide of SEQ ID NO:3 or SEQ ID NO:6).

A further aspect of the present invention is a method of amelioratingsymptoms associated with CNS ischemia in a subject, by administering acompound that binds to glial or microglial cells at the LRP/α2M receptor(the receptor bound by a peptide of SEQ ID NO:3 or SEQ ID NO:6) in atreatment effective amount.

A further aspect of the present invention is a method of treatingcerebral ischemia or inflammation of the CNS by administering a LRP/α2Mreceptor ligand, such as peptide comprising SEQ ID NO:3 or SEQ ID NO:6.

A further aspect of the present invention is a therapeutic peptide ofSEQ ID NO: 3, or a dimer of two peptides wherein each peptide comprisesSEQ ID NO:2, or a peptide of SEQ ID NO:6, and pharmaceuticalcompositions thereof.

A further aspect of the present invention is a method of screening acompound for the ability to suppress glial or microglial activation byincubating an activated glial or microglial cell culture with thecompound, and then measuring a marker of microglial activation such asnitric oxide.

A further aspect of the present invention is a method of screening acompound for the ability to suppress glial or microglial activation, bypre-incubating a glial or microglial cell culture with the compound;incubating the cell culture with a known activator of glia or microglia;and then measuring a marker of glial or microglial activation.

A further aspect of the present invention is a method of screening atest compound for the ability to suppress glial or microglialactivation, by determining whether the compound binds to glia ormicroglia at the same receptor to which peptides of SEQ ID NO:3 or SEQID NO:6 bind (that is, the LRP/α2M receptor).

A further aspect of the present invention is a method of suppressingmacrophage activation in a mammalian subject, by administering acompound that binds to macrophage cells at the LRP/α2M receptor (thereceptor bound by a peptide of SEQ ID NO:3 or SEQ ID NO:6).

A further aspect of the present invention is a method of treatingatherosclerosis or of reducing the formation of atherosclerotic plaques,comprising administering a compound that binds to macrophage cells atthe LRP/α2M receptor (the receptor bound by a peptide of SEQ ID NO:3 orSEQ ID NO:6).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphs the production of nitrite by cultures of glial cells fromApoE-deficient mice (solid bar), ApoE3transgenic mice (hatched bar), andcontrol mice (white bar), after exposure to lipopolysaccharide (LPS).Responses were measured at 24 and 60 hours after stimulation of cellcultures by LPS.

FIG. 2 graphs nitrite production by enriched microglia primary culturesfrom ApoE-deficient mice after stimulation with LPS and subsequentaddition of peptides of SEQ ID NO:3 (tandem repeat peptides). Peptideswere added in doses of from 0 μM to 1000 μM, and a dose dependentdecrease in nitrite production was observed. As a control, peptides ofSEQ ID NO:2 were added to cultures (solid bar); no decrease in nitriteproduction was observed.

FIG. 3A graphs intracellular calcium content over time in murineperitoneal macrophages, after exposure to either ApoE3 (squares) orApoE4 (circles).

FIG. 3B graphs inositol trisphosphate (IP3) in murine peritonealmacrophages exposed to either ApoE3 (squares) or ApoE4 (circles). Thegraph shows the percent change in IP3 content in treated cells comparedto control cells exposed to vehicle but not ApoE.

FIG. 4 graphs production of TNFα(picogram/ml) by microglia primarycultures from ApoE-deficient mice after addition of peptides of SEQ IDNO:6 (squares), or addition of peptides of SEQ ID NO:6 and LPS (100ng/ml) (circles). Peptides were added in doses of 10 μM, 100 μM and 1000μM.

FIG. 5 is a graph of the optical density of cell cultures, as a measureof cell viability. Cultures of microglia from ApoE-deficient mice wereexposed to either peptides of SEQ ID NO:6 (squares), or peptides of SEQID NO:6 and LPS (100 ng/mil) (circles). Peptides were added in doses of10 μM, 100 μM and 1000 μM.

FIG. 6 graphs production of TNFα(picogram/ml) by microglia primarycultures from ApoE-deficient mice after addition of peptides of SEQ IDNO:6 (squares), or addition of peptides of SEQ ID NO:6 and LPS (100ng/ml) (circles). Peptides were added in doses of 1 μM, 10 μM, 100 μMand 1000 μM.

FIG. 7 is a graph of the optical density of cell cultures, as a measureof cell viability. Cultures of microglia from ApoE-deficient mice wereexposed to either peptides of SEQ ID NO:6 (squares), or peptides of SEQID NO:6 and LPS (100 ng/ml) (circles). Peptides were added in doses of10 μM, 100 μM and 1000 μM.

FIG. 8. Changes in [Ca²⁺]_(i) in macrophages treated with apoE. Panel A:Changes in [Ca²⁺]_(i) in a single Fura-2/AM loaded peritoneal macrophageon stimulation with apoE (100 pM). Details for measuring [Ca²⁺]_(i) aredescribed in the Examples below. The graph shown is representative of 5individual experiments using 20-30 cells each. Approximately 70-80% ofthe macrophage demonstrated changes in [Ca²⁺]_(i) upon stimulation withapoE. The arrow indicates the time of addition of apoE. Panel B: Effectof apoE concentration on changes in [Ca²⁺]_(i). The changes in[Ca²⁺]_(i) in individual cells were measured prior to and followingexposure to varying concentrations of apoE. The data are displayed asmean (S.E. and are representative of two independent experiments; ineach case 25-30 cells were analyzed cells per study.

FIG. 9. Changes in IP₃ in macrophages treated with apoE. Panel A: Effectof apoE on IP₃ synthesis in macrophages, and modulation by pertussistoxin. These results are representative of two independent experimentsperformed in duplicate and expressed as % change in IP₃ formation atdifferent time periods in myo-[2-³H] inositol-labeled cells stimulatedwith apoE (100 pM) in the presence (open circles) and absence (filledcircles) of pertussis toxin. Panel B: Effect of apoE concentration onIP₃ formation in [³H] labeled macrophages. The cells were stimulatedwith varying concentrations of apoE for 60s and IP₃ determined. Resultsare displayed as mean (S.E. and are representative of two individualexperiments performed in duplicate.

FIG. 10A shows the performance of mice with and without treatment onrotorod latency after closed head injury.

FIG. 10B shows the weight gain of mice with and without treatment afterclosed head injury.

FIG. 10C shows the performance of mice with and without treatment in awater maze latency test after closed head injury.

FIG. 10D shows the survival of mice with and without treatment afterclosed head injury.

DETAILED DESCRIPTION OF THE INVENTION

The LRP/(α2M receptor.

The LRP/α2M receptor is known. In overview, following modification bylipoprotein lipase and the association of apolipoproteins, very largedensity lipoproteins (VLDL) and chylomicron become remnants, and arecleared hepatically by a receptor-mediated mechanism. Althoughrecognized as distinct from the low density lipoprotein (LDL) receptor,the remnant receptor also has a high affinity for apolipoprotein E, andrecognizes the remnant particles via incorporated apoE moieties. In1988, this remnant receptor was cloned, and dubbed the LDLreceptor-related related protein, or “LRP”. The LRP is a large receptor,with a primary sequence of 4525 amino acids, and bears many structuralsimilarities to other members of the LDL receptor family. Like the LDLreceptor, the extracellular domain of LRP includes a cysteine-enrichedligand binding domain and EGF precursor homology domain which arebelieved to play a role in the acid-dependent dissociation of ligandfrom the receptor. Unlike the LDL receptor, however, the O-lined sugardomain is not present in the extracellular portion adjacent to themembrane. As with all of the members of the LDL receptor family, LRP isa transmembrane protein, and is anchored by a single transmembranesegment. The cytoplasmic tail of the protein is 100 amino acids,approximately twice as long as the LDL receptor, and contains the NP×Ymotif, which is believed to be necessary for targeted coated-pitmediated endocytosis (See, e.g., Krieger M. Herz J. Structures andfunctions of multiligand lipoprotein receptors: macrophage scavengerreceptors and LDL receptor-related protein (LRP). Annual Review ofBiochemistry. 63:601-37, 1994. UI: 95069975; Misra UK. Chu CT. Gawdi G.Pizzo SV. The relationship between low density lipoprotein-relatedprotein/alpha 2-macroglobulin (alpha 2M) receptors and the newlydescribed alpha 2M signaling receptor. Journal of Biological Chemistry.269(28):18303-6, 1994).

The ApoE genotype in humans has been correlated with outcome in avariety of acute neurological conditions including cerebral hemorrhage,closed head injury, stroke and cognitive deterioration aftercardiopulmonary bypass. See, e.g., Seliger et al., Neurology (Abstract)page A213 (1997); Alberts et al., Stroke 27:183 (abstract)(1996);Connolly et al., Stroke 27:174 (abstract) (1996); Sorbi et al.,Neurology 46:A307 (abstract) (1996); Newman et al., Ann. Thorac. Surg.59:1326 (1995). ApoE is the primarily apolipoprotein produced in thecentral nervous system (CNS) and is upregulated after injury. Laskowitzet al., J. Neuroimmunol. 76:70 (1997).

ApoE has been demonstrated to have immunomodulatory effects in vitro,including suppression of lymphocyte proliferation and immunoglobulinsynthesis after mitogenic challenge. Avila et al., J. Biol. Chem.257:5900 (1982); Edgington and Curtiss, Cancer Res. 41:3786 (1981). ApoEis secreted in large quantities by macrophage after peripheral nerveinjury, and by astrocytes and oligodendrocytes after CNS injury. Stollet al., Glia 2:170 (1989); Stoll and Mueller, Neurosci. Lett. 72:233(1986).

Apolipoprotein E binds to the low-density lipoprotein (LDL) receptor, aswell as to the LDL receptor-related protein (LRP). The region of ApoEthat is involved in receptor interaction is in the vicinity of aminoacid residues 135-160, and is rich in basic amino acids includingarginine and lysine. This interaction of apolipoprotein E and the LDLreceptor is important in lipoprotein metabolism. In studies of the LDLreceptor-binding activity of apolipoprotein E, it is typically complexedwith phospholipid. The protein has been described as essentiallyinactive in the lipid-free state. Innerarity et al., J. Biol. Chem.254:41864190 (1979).

Various amino acid substitutions in the receptor binding region of ApoEhave been studied for their effects on ApoE-LDL receptor binding.Substitution of either arginine or lysine at residues 136, 142, 145 and146 with neutral residues decreased normal apoE3 binding activity.Weisgraber, Advances in Protein Chemistry 45:249 (1994); Lalazar et al.,J. Biol. Chem. 263:3542 (1988). No single substitution of a basicresidue within the receptor-binding region of ApoE3 completely disruptsLDL receptor binding, suggesting that no one residue is critical forthis interaction. It has been postulated that regions of ApoE outsidethe LDL binding region are necessary to maintain the receptor-bindingregion in an active binding conformation. Weisgraber, Advances inProtein Chemistry 45:249 (1994). Dyer et al., J. Biol. Chem. 266:15009(1991), studied lipid-free synthetic peptide fragments comprisingresidues 141-155 of ApoE, and a dimeric peptide of this sequence. Nobinding activity was observed with the monomer of this peptide; lowlevels of binding were observed with the dimer (˜1% of LDL activity).

Several receptors that bind ApoE with high affinity have beenidentified, including the scavenger receptor, VLDL receptor, LDLreceptor, and LRP receptors. These three receptors have areas of highsequence similarity. The scavenger receptor is known to be present onmicroglia, and preferentially binds acytylated and oxidized LDL. Thescavenger receptor may be particularly relevant under inflammatory(oxidizing) conditions. Scavenger receptors are also known to beupregulated in microglia after injury. LRP receptors are known to bepresent on macrophages.

The microglia is the primary immunocompetent cell in the central nervoussystem. Acute CNS insult, as well as chronic conditions such as HWencephalopathy, epilepsy, and Alzheimer's disease (AD) are associatedwith microglial activation. McGeer et al., Glia 7:88 (1993); Rothwelland Relton, Cerebrovasc. Brain Metab. Rev. 5:178 (1993); Giulian et al.,J. Neuroscience, 16:3139 (1996); Sheng et al., J. Neurochem 63:1872(1994). Microglial activation results in the production of nitric oxide(NO) and other free radical species, and the release of proteases,inflammatory cytokines (including IL-1 β, IL-6 and TNFα), and aneurotoxin that works through the NMDA receptor. Giulian et al., J.Neuroscience, 16:3139 (1996). Microglial activation can be assessed bymeasuring the production of nitrite, a stable product of nitric oxideformation. See, e.g, Barger and Harmon, Nature 388:878 (1997).

The present inventors determined that apoE modulates the activation ofglia in the CNS, and further identified a peptide that suppresses theactivation of microglia. While not wishing to be bound to a singletheory, the present inventors hypothesized that ApoE binding to amicroglial receptor affects the phenotype of the microglia, decreasingthe responsiveness of the microglia to various activators, and thereforedecreasing the release of inflammatory compounds from the microglia thatwould otherwise occur in the presence of such activators. The ApoE maybe binding to the same receptor as is bound by the activating compounds,or may be binding to a receptor independent from that bound byactivators. In lymphocytes, ApoE has been shown to block activation by avariety of compounds, including LPS, the lectin PHA, and anti-CD3antibody; these activators are known to bind to distinct receptors onlymphocytes. The methods and compounds of the present invention aredesigned to prevent or suppress the receptor-mediated activation ofmicroglia, and thus prevent or reduce the deleterious neurologicaleffects associated with activated microglia. Peptides and othertherapeutic molecules according to the present invention are able tobind to receptors on glia, and decrease the responsiveness of the cellto various activators. In this manner, methods and compounds accordingto the present invention may be used to treat, ameliorate, or preventcertain signs, symptoms, and/or deleterious neurological effects ofacute and/or chronic CNS injury. The effect of the present methods andcompounds may be assessed at the cellular or tissue level (e.g.,histologically or morphometrically), or by assessing a subject'sneurological status. Methods of assessing a subject's neurologicalstatus are known in the art.

Laskowitz et al., J. Neuroimmunology 76:70 (June 1997) describedexperiments in which mixed neuronal-glial cell cultures fromapoE-deficient mice were stimulated with lipopolysaccharide (LPS). Itwas found that preincubation of the cell cultures with apoE blockedglial secretion of TNFα in a dose-dependent manner. Laskowitz et al., J.Cerebral Blood Flow and Metabolism, 17:753-758 (July 1997) compared theneurologic and histologic outcome of ApoE-deficient mice subjected toocclusion of the cerebral artery for either 60 or 90 minutes, with arecovery period of 24 hours. When subjected to 60 minutes of occlusion,ApoE-deficient mice were reported to have larger infarcts and moresevere hemiparesis than wild-type mice. In mice subjected to 90 minutesof occlusion, mortality was 40% in ApoE-deficient mice compared to 0% inwild-type mice.

Barger S W and Harmon A D, Nature 388:878 (August 1997) reported thattreatment of microglia with a secreted derivative of beta-amyloidprecursor protein (sAPP-alpha) activated microglia, induced inflammatoryreactions in microglia, and enhanced the production of neurotoxins bymicroglia. The ability of sAPP-alpha to activate microglia was blockedby prior incubation of the sAPP-alpha protein with apolipoprotein E3 butnot apolipoprotein E4.

Suitable subjects for carrying out the methods of the present inventioninclude male and female mammalian subjects, including humans, non-humanprimates, and non-primate mammals. Subjects include veterinary(companion animal) subjects, as well as livestock and exotic species.

The present methods and compounds are useful in preventing, treating, orameliorating neurological signs and symptoms associated with acute CNSinjury; as used herein, acute CNS injury includes but is not limited tostroke (caused by thrombosis, embolism or vasoconstriction), closed headinjury, global cerebral ischemia (e.g., ischemia due to systemichypotension of any cause, including cardiac infarction, cardiacarrhythmia, hemorrhagic shock, and post coronary artery bypass graftbrain injury) and intracranial hemorrhage. Further, the present methodsand compounds are useful in preventing, treating, or amelioratingneurological signs and symptoms associated with chronic neurologicaldisease, including but not limited to Alzheimer's disease (AD) andHIV-associated encephalopathy. The present methods and compounds arealso useful in preventing, treating, or ameliorating the neurologicalsigns and symptoms associated with inflammatory conditions affecting thenervous system including the CNS, including but not limited to multiplesclerosis, vasculitis, acute disseminated encephalomyelitis, andGuillain-Barre syndrome.

Stated in a different way, the present methods and compounds are usefulin preventing, suppressing or reducing the activation of glia in the CNSthat occurs as a part of acute or chronic CNS disease. The suppressionor reduction of glial activation can be assessed by various methods aswould be apparent to those in the art; one such method is to measure theproduction or presence of compounds that are known to be produced byactivated glia, and compare such measurements to levels of the samecompounds in control situations. Alternatively, the effects of thepresent methods and compounds in suppressing, reducing or preventingmicroglial activation may be assessed by comparing the signs and/orsymptoms of CNS disease in treated and control subjects, where suchsigns and/or symptoms are associated with or secondary to activation ofmicroglia.

Ischemic damage to the central nervous system may result from eitherglobal or focal ischemic conditions. Global ischemia occurs where bloodflow to the entire brain ceases for a period of time, such as duringcardiac arrest. Focal ischemia occurs when a portion of the brain isdeprived of normal blood flow, such as during thromboembolytic occlusionof a cerebral vessel, traumatic head injury, edema and brain tumors.Much of the CNS damage due to cerebral ischemia occurs during the hoursor even days following the ischemic condition, and is secondary to therelease of cytotoxic products by damaged tissue.

In Alzheimer's disease, studies indicate that anti-inflammatory drugsmay delay the onset or progression of the disease. Breitner et al.,Neurobiol. Aging 16:523 (1995); Rogers et al., Neurology 43:1609 (1993).Microglia express markers of activation in AD, suggesting that crucialinflammatory events in AD involve microglia. Such activated microgliacluster near amyloid plaques. Griffm et al., J. Neuropath. Exp. Neurol.54:276 (1995). Microglia are also activated in epilepsy (see Sheng etal., J. Neurochem 63:1872 (1994).

In subjects with head injuries, AD-like changes are synergistic withApoE genotype. The ApoE4 allele has been associated with the extent ofamyloid β-protein deposition following head injury. Mayeux et al.,Neurology 45:555 (1995); Nicoll et al., Nat. Med. 1:135 (1995).

As used herein, the terms “combating”, “treating” and “ameliorating” arenot necessarily meant to indicate a reversal or cessation of the diseaseprocess underlying the CNS condition afflicting the subject beingtreated. Such terms indicate that the deleterious signs and/or symptomsassociated with the condition being treated are lessened or reduced, orthe rate of progression is reduced, compared to that which would occurin the absence of treatment. A change in a disease sign or symptom maybe assessed at the level of the subject (e.g., the function or conditionof the subject is assessed), or at a tissue or cellular level (e.g., theproduction of markers of glial activation is lessened or reduced). Wherethe methods of the present invention are used to treat chronic CNSconditions (such as Alzheimer's disease), the methods may slow or delaythe onset of symptoms such as dementia, while not necessarily affectingor reversing the underlying disease process.

Active Compounds.

Active compounds that may be used to carry out the present inventioninclude ligands or agonists that specifically and/or selectively bind tothe LRP/α2M receptor. Examples of such compounds include, but are notlimited to, 1) alpha 2 macroglobulin; 2) pseudomonas exotoxin; 3)lipoprotein lipase; 4) apolipoprotein E; 5) oxidized and/or acetylatedLDL; 6) receptor associated protein (RAP); 7) remnant particles; 8) lowdensity lipoprotein (LDL); 9) high denity lipoprotein (HDL); 10)lactoferrin; 11) tissue plasminogen activator (tPA); 12) urineplasminogen activator (uPA); etc.

Amino acid residues 100-200 of each isoform of the ApoE moleculecomprise the ApoE receptor binding region. More specifically, thereceptor binding region of ApoE is within amino acid residues 130-160 ofeach isoform of the ApoE molecule (SEQ ID NO:4 and SEQ ID NO:5), andmore specifically is within amino acid residues 140-155 (HLRKLRKRLLRDADDL) (SEQ ID NO:1). See, e.g., Weisgraber, Apolipoprotein E:Structure-Function Relationships, Advances in Protein Chemistry 45:249(1994). The amino acid interchanges that define the E2, E3 and E4isoforms are not found within the region of amino acid residues 140-155,but do influence the overall structure of the apolipoprotein molecule.ApoE2 and ApoE3 molecules form covalently bound homodimers; ApoE4molecules do not.

As used herein, the term homodimer refers to a molecule composed of twomolecules of the same chemical composition; the term heterodimer refersto a molecule composed of two molecules of differing chemicalcomposition.

The present inventors utilized a 9-mer monomer having an amino acidsequence LRKLRKRLL (SEQ ID NO:2). This 9 amino acid sequence is foundwithin the larger ApoE receptor binding sequence region identifiedabove, and is found at amino acid positions 141-149 of ApoE. The presentinventors constructed a dimer of SEQ ID NO:2, i.e., a peptide having anamino acid sequence of LRKLRKRLL LRKLRKRLL (SEQ ID NO:3). Peptides ofSEQ ID NO:3 suppressed microglial activation in a dose-dependentfashion. Use of the monomer (monomer peptides of SEQ ID NO:2) did notsuppress microglial activation. (See FIG. 2).

The present inventors further utilized a 20-mer monomer having an aminoacid sequence TEELRVRLAS HLRKLRKRLL (SEQ ID NO:6). This 20 amino acidsequence is found at amino acid positions 130-149 of ApoE, and comprisesthe 9-mer SEQ ID NO:2. Peptides of SEQ ID NO:6 suppressed microglialactivation in a dose-dependent fashion (see FIGS. 4-7).

Clay et al., Biochemistry 34:11142 (1995) reported that dimeric peptidesof amino acids 141-155 or 141-149 were both cytostatic and cytotoxic toT lymphocytes in culture. Cardin et al. Biochem Biophys Res. Commun.154:741 (1988) reported that a peptide of apoE 141-155 inhibited theproliferation of lymphocytes. A peptide consisting of a tandem repeat ofamino acids 141-155, as well as longer monomeric peptides comprising the141-155 region, was found to cause extensive and specific degenerationof neurites from embryonic chicks in vitro. Crutcher et al., Exp.Neurol. 130:120 (1994). These authors suggested that peptide sequencesassociated with apoE might contribute directly to neurodegenerativeprocesses.

Peptides of the present invention may be produced by standard techniquesas are known in the art.

Active compounds (or “active agents”) useful in the methods of thepresent invention include those that compete with a peptide of SEQ IDNO:3, and/or a peptide of SEQ ID NO:6 in binding to microglial receptorsto thereby prevent or suppress activation of the microglia by moleculesthat would otherwise activate microglia.

Peptides useful in the present methods include those comprising the ApoELDL receptor binding sequence (including multiple repeats thereof,including but not limited to dimers and trimers); and conjugates of twoor more peptides, each of which comprises a peptide as described hereinor a peptide comprising the LDL receptor binding sequence. One ApoEreceptor binding sequence is provided in SEQ ID NO:1. A preferredpeptide comprises or consists of multiple repeats of SEQ ID NO:2,preferably dimers thereof. Thus, a preferred peptide useful in thepresent methods is SEQ ID NO:3 (a tandem repeat of LRKLRKRLL), orpeptides comprising SEQ ID NO:3. Further preferred peptides comprise orconsist of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

The ability of a linear tandem repeat of amino acids 141-155 (the141-155 dimer) to bind the LDL receptor was studied by Dyer et al., J.Lipid Research 36:80 (1995). A series of modified peptides wasconstructed and assessed for LDL binding ability. These authors reportthat deletion of the charged amino terminal residues (including arg142and lys43) in 145-155 or 144-150 dimers abolished the LDL receptoractivities of the peptides. These authors conclude that LDL-receptorbinding activity of the 141-155 dimer is dependent on at least twoclusters of basic amino acids present on the hydrophilic face of theamphipathic alpha-helix of the 141-155, 141-150, 141-155 (1lys143-->ala)and 141-155 (arg150-->ala) dimer peptides. Dyer et al., J. Biol. Chem.266:15009 (1991) reported that a self-conjugate of peptide 141-155, anda peptide consisting of a tandem repeat of 141-155, were able to inhibitboth lymphocyte proliferation and ovarian androgen production. Dyer etal., J. Biol. Chem. 266:22803 (1991) investigated the LDL bindingability of a dimeric 141-155 tandem peptide, and a trimeric 141-155peptide. Binding was decreased with amino acid substitutions ofLys-143--->Ala, Leul144--->Pro, and Argl150--->Ala.

Lalazar et al., J. Biol. Chem. 263:3542 (1988) investigated variants ofApoE for binding to the LDL receptor. When neutral amino acids weresubstituted for basic residues at positions 136, 140, 143, and 150,binding activity was reduced. Where proline was substituted for leucinel144 or alanine 152, binding was reduced. However, slightly enhancedreceptor binding was displayed by a variant in which arginine wassubstituted for serine139 and alanine was substituted for leucine 149.

Compounds that are useful in the present method include those which actas antagonists for the microglial receptor bound by peptides of SEQ IDNO:3 and/or SEQ ID NO:6. Antibodies that selectively target and bind tothis receptor can also be used as antagonists of microglial activationaccording to the present invention. Such antibodies selectively orspecifically bind to the receptor bound by peptides of SEQ ID NO:3and/or peptides of SEQ ID NO:6.

Peptides of SEQ ID NO:3, SEQ ID NO:6, or conformational analoguesthereof, are an aspect of the present invention. Such compounds arepeptides or peptidomimetics having a core sequence of amino acids with aconformation in aqueous solution that interacts with receptor moleculeson glial cells to block the activation of glial cells that wouldotherwise occur in conjunction with acute or chronic CNS injury, orexposure to known activators of microglia such as LPS. Stated anotherway, such compounds are characterized by the ability to compete withpeptides of SEQ ID NO:3 and/or peptides of SEQ ID NO:6 for binding tomicroglia, and by their ability to suppress microglial activation byknown activators such as LPS.

Another variation of the therapeutic peptides of the present inventionis the linking of from one to five amino acids or analogues to theN-terminal or C-terminal amino acid of the therapeutic peptide. Analogsof the peptides of the present invention may also be prepared by addingfrom one to five additional amino acids to the N-terminal, C-terminal,or both N- and C-terminals, of an active peptide, where such amino acidadditions do not adversely affect the ability of the peptide to bind tomicroglia at the site bound by a peptide of SEQ ID NO:3 and/or SEQ IDNO:6.

Changes in the amino acid sequence of peptides can be guided by knownsimilarities among amino acids and other molecules or substituents inphysical features such as charge density, hydrophobicity,hydrophilicity, size and configuration, etc. For example, the amino acidThr may be replaced by Ser and vice versa, and Leu may be replaced byIle and vice versa. Further, the selection of analogs may be made bymass screening techniques known to those skilled in the art (e.g.,screening for compounds which bind to microglia at the receptor bound bya peptide of SEQ ID NO:3 and/or SEQ ID NO:6). A preferred exchange is toreplace Ser with Arg, to increase the arginine content of the peptide;examples include peptides of or comprising SEQ ID NO:7, SEQ ID NO:8 orSEQ ID NO:9. A further preferred exchange is to substitute alanine forleucine 149.

Peptides of the present invention may also be characterized as shortpeptides of from about 20 amino acids, 22 amino acids, 24 amino acids,26 amino acids, 28 amino acids, 30 amino acids, 35 amino acids, or 40amino acids, up to about 22 amino acids, 24 amino acids, 26 amino acids,28 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 aminoacids, 50amino acids or more, where the peptides comprise the 18-aminoacid sequence LRKLRKRLL LRKLRKRLL (SEQ ID NO:3), or variants thereofthat retain the receptor binding ability of peptides of SEQ ID NO:3. Apreferred peptide useful in the present invention is one consisting ofor comprising SEQ ID NO:3. Where longer peptides are employed, thoseincorporating amino acid sequences derived from the ApoE sequenceimmediately surrounding amino acid residues 141-149 are preferred. Wherepeptides longer than 18 amino acids are employed, it is contemplatedthat they may include virtually any other amino acid sequences so longas the resultant peptide maintains its ability to bind to microglial andsuppress microglia activation in acute and chronic CNS inflammation. Thepresent invention includes those variations of the ApoE sequence at141-149 which are known to retain the ability LDL receptor-bindingability. Synthetic peptides may further be employed, for example, usingone or more D-amino acids in place of L-amino acids, or by adding groupsto the N- or C-termini, such as by acylation or amination.

Peptides of the present invention may also be characterized as shortpeptides of from about 10 amino acids, 12 amino acids, 14 amino acids,15 amino acids, 18 amino acids, 20 amino acids, 22 amino acids, 24 aminoacids, 26 amino acids, 28 amino acids, 30 amino acids, 35 amino acids,or 40 amino acids, up to about 15 amion acids, 22 amino acids, 24 aminoacids, 26 amino acids, 28 amino acids, 30 amino acids, 35 amino acids,40 amino acids, 45 amino acids, 50 amino acids or more, where thepeptides comprise the 9-amino acid sequence LRKLRKRLL (SEQ ID NO:2), orvariants thereof that retain the receptor binding ability of peptides ofSEQ ID NO:3 and/or SEQ ID NO:6. A preferred peptide useful in thepresent invention is one consisting of or comprising the apoE receptorbinding region; a particularly preferred peptide consists of orcomprises SEQ ID NO:6. Where longer peptides are employed, thoseincorporating amino acid sequences derived from within the apoE receptorbinding regtion, or the ApoE sequence immediately surrounding the apoEreceptor binding region, are preferred, although it is contemplated thatthese peptides may include virtually any other amino acid sequences solong as the resultant peptide maintains its ability to bind to microgliaand suppress microglia activation in acute and chronic CNS inflammation.The present invention includes those variations of the ApoE sequence at141-149 which are known to retain the ability LDL receptor-bindingability. Synthetic peptides may further be employed, for example, usingone or more D-amino acids in place of L-amino acids, or by adding groupsto the N- or C-termini, such as by acylation or amination.

The peptides of the present invention include not only natural aminoacid sequences, but also peptides which are analogs, chemicalderivatives, or salts thereof. The term “analog” or “conservativevariation” refers to any polypeptide having a substantially identicalamino acid sequence to the therapeutic peptides identified herein, andin which one or more amino acids have been substituted with chemicallysimilar amino acids. For example, a polar amino acid such as glycine orserine may be substituted for another polar amino acid; a basic aminoacid may be substituted for another basic amino acid, or an acidic aminoacid may be substituted for another acidic amino acid; or a non-polaramino acid may be substituted for another non-polar amino acid. Thereterm “analog” or “conservative variation” as used herein also refers toa peptide which has had one or more amino acids deleted or added to apolypeptide of the present invention, but which retains a substantialsequence similarity (at least about 85% sequence similarity, andpreferably at least 90%, 92%, 94%, 95%, 96%, 98% or even 99% sequencesimilarity), where the peptide retains the ability to suppressmicroglial activation as described herein.

The amino acids constituting peptides of the present invention may be ofeither the L-configuration or the D-configuration. Therapeutic peptidesof the present invention may be in free form or the form of a salt,where the salt is pharmaceutically acceptable.

As used herein, the term “administering to the brain of a subject”refers to the use of routes of administration, as are known in the art,that provide the compound to the central nervous system tissues, and inparticular the brain, of a subject being treated.

Preferably, the compounds of the present invention are used incombination with a pharmaceutically acceptable carrier. The presentinvention thus also provides pharmaceutical compositions suitable foradministration to mammalian subjects. Such compositions comprise aneffective amount of the compound of the present invention in combinationwith a pharmaceutically acceptable carrier. The carrier may be a liquid,so that the composition is adapted for parenteral administration, or maybe solid, i.e., a tablet or pill formulated for oral administration.Further, the carrier may be in the form of a nebulizable liquid or solidso that the composition is adapted for inhalation. When administeredparenterally, the composition should by pyrogen free and in anacceptable parenteral carrier. Active compounds may alternatively beformulated encapsulated in liposomes, using known methods. Additionally,the intranasal administration of peptides to treat CNS conditions isknown in the art (see, e.g., U.S. Pat. No. 5,567,682 to Pert, regardingintranasal administration of peptide T to treat AD). (All patentsreferenced herein are intended to be incorporated by reference herein intheir entirety.) Preparation of a compound of the present invention forintranasal administration may be carried out using techniques as areknown in the art.

Pharmaceutical preparations of the compounds of the present inventionmay optionally include a pharmaceutically acceptable diluent orexcipient.

An effective amount of the compound of the present invention is thatamount that decreases microglial activation compared to that which wouldoccur in the absence of the compound; in other words, an amount thatdecreases the production of neurotoxic compounds by the microglia,compared to that which would occur in the absence of the compound. Theeffective amount (and the manner of administration) will be determinedon an individual basis and will be based on the specific therapeuticmolecule being used and a consideration of the subject (size, age,general health), the condition being treated (AD, acute head injury,cerebral inflammation, etc.), the severity of the symptoms to betreated, the result sought, the specific carrier or pharmaceuticalformulation being used, the route of administration, and other factorsas would be apparent to those skilled in the art. The effective amountcan be determined by one of ordinary skill in the art using techniquesas are known in the art. Therapeutically effective amounts of thecompounds described herein may be determined using in vitro tests,animal models or other dose-response studies, as are known in the art.

The compounds of the present invention may be administered acutely(i.e., during the onset or shortly after events leading to cerebralinflammation or ischemia), or may be administered prophylactically(e.g., before scheduled surgery, or before the appearance of neurologicsigns or symptoms), or administered during the course of a degenerativedisease to reduce or ameliorate the progression of symptoms that wouldotherwise occur. The timing and interval of administration is variedaccording to the subject's symptoms, and may be administered at aninterval of several hours to several days, over a time course of hours,days, weeks or longer, as would be determined by one skilled in the art.

The typical daily regime may be from about .01 μg/kg body weight perday, from about 10 μg/kg body weight per day, from about 100 μg/kg bodyweight per day, from about 1000 μg/kg body weight per day, from about10,000 μg/kg body weight per day, from about 100,000 μg/kg body weightper day.

The blood-brain barrier presents a barrier to the passive diffusion ofsubstances from the bloodstream into various regions of the CNS.However, active transport of certain agents is known to occur in eitherdirection across the blood-brain barrier. Substances that may havelimited access to the brain from the bloodstream can be injecteddirectly into the cerebrospinal fluid. Cerebral ischemia andinflammation are also known to modify the blood-brain barrier and resultin increased access to substances in the bloodstream.

Administration of a compound directly to the brain is known in the art.Intrathecal injection administers agents directly to the brainventricles and the spinal fluid. Surgically-implantable infusion pumpsare available to provide sustained administration of agents directlyinto the spinal fluid. Lumbar puncture with injection of apharmaceutical compound into the cerebrospinal fluid (“spinalinjection”) is known in the art, and is suited for administration of thepresent compounds.

Pharmacologic-based procedures are also known in the art forcircumventing the blood brain barrier, including the conversion ofhydrophilic compounds into lipid-soluble drugs. The active agent may beencapsulated in a lipid vesicle or liposome.

The intra-arterial infusion of hypertonic substances to transiently openthe blood-brain barrier and allow passage of hydrophilic drugs into thebrain is also known in the art. U.S. Pat. No. 5,686,416 to Kozarich etal. discloses the co-administration of receptor mediated permeabilizer(RMP) peptides with compounds to be delivered to the interstitial fluidcompartment of the brain, to cause an increase in the permeability ofthe blood-brain barrier and effect increased delivery of the compoundsto the brain. Intravenous or intraperitoneal administration may also beused to administer the compounds of the present invention.

One method of transporting an active agent across the blood-brainbarrier is to couple or conjugate the active agent to a second molecule(a “carrier”), which is a peptide or non-proteinaceous moiety selectedfor its ability to penetrate the blood-brain barrier and transport theactive agent across the blood-brain barrier. Examples of suitablecarriers include pyridinium, fatty acids, inositol, cholesterol, andglucose derivatives. The carrier may be a compound which enters thebrain through a specific transport system in brain endothelial cells.Chimeric peptides adapted for delivering neuropharmaceutical agents intothe brain by receptor-mediated transcytosis through the blood-brainbarrier are disclosed in U.S. Pat. No. 4,902,505 to Pardridge et al.These chimeric peptides comprise a pharmaceutical agent conjugated witha transportable peptide capable of crossing the blood-brain barrier bytranscytosis. Specific transportable peptides disclosed by Pardridge etal. include histone, insulin, transferrin, and others. Conjugates of acompound with a carrier molecule, to cross the blood-brain barrier, arealso disclosed in U.S. Pat. No. 5,604,198 to Poduslo et al. Specificcarrier molecules disclosed include hemoglobin, lysozyme, cytochrome c,ceruloplasmin, calmodulin, ubiquitin and substance P. See also U.S. Pat.No. 5,017,566 to Bodor.

An alternative method of administering peptides of the present inventionis carried out by administering to the subject a vector carrying anucleic acid sequence encoding the peptide, where the vector is capableof entering brain cells so that the peptide is expressed and secreted,and is thus available to microglial cells. Suitable vectors aretypically viral vectors, including DNA viruses, RNA viruses, andretroviruses. Techniques for utilizing vector deliver systems andcarrying out gene therapy are known in the art. Herpesvirus vectors area particular type of vector that may be employed in administeringcompounds of the present invention.

Screening Methods.

Also disclosed herein are methods of screening compounds for the abilityto prevent or reduce microglial activation under conditions of cerebralischemia or cerebral inflammation. Such methods comprise contacting anactivated microglial cell with a test compound, and detecting whetherthe test compound binds to microglia at the same receptor at whichpeptides of SEQ ID NO:3 and/or SEQ ID NO:6 bind. The contacting step maybe carried out in vitro, for example in cell culture. A competitivebinding assay may be used to detect whether the test compound binds tothe same receptor that is bound by peptides of SEQ ID NO:3 and/or SEQ IDNO:6.

An additional method of screening a test compound for the ability tosuppress microglial activation comprises incubating an activatedmicroglial cell culture with a test compound, and measuring at least onemarker of microglial activation. A decrease in a marker of microglialactivation (compared to the level of that marker that would occur in theabsence of the test compound) indicates that the test compound is ableto suppress, prevent or reduce microglial activation. An exemplarymarker of microglial activation is the production of nitric oxide.

A further method of screening a test compound for the ability tosuppress microglial activation involves pre-incubating a microglial cellculture with a test compound, then incubating the microglial cellculture with a compound that is known to activate microglia. At leastone marker of microglial activation is then measured, and a decrease inthe activation marker (compared to that which occurs in the absence ofthe pre-incubation step) indicates that the test compound is able toaffect microglial activation. An exemplary marker of microglialactivation is the production of nitric oxide.

Atherosclerosis.

It known that the inflammatory process mediates an aspect of theatherosclerotic process. See, e.g., Hansson, Basic Res. Cardiol.,89(1):41 (1994); Berliner et al., Circulation 91:2488 (1995); Watanabeet al., Int. J. Cardiol. 54:551 (1997). ApoE is known to be secreted bymacrophages locally at blood vessel walls (although the amount secretedby macrophages in an individual is trivial compared to the amount ofApoE produced by the liver). In the classic model of atherosclerosis,ApoE functions to remove cholesterol from the blood stream and deliverit to macrophages or to the liver. However, it has become apparent thatApoE secreted by macrophages at the blood vessel wall decreasesatherosclerotic plaque formation, independent of any lipid metabolismeffects. ApoE-deficient mice are accepted as a model ofhypercholesteremia and atherosclerotic disease; providing ApoE-secretingmacrophages to such mice dramatically decreases atherosclerotic plaqueformation. Linton et al., Science, 267:1034 (1995). Conversely,replacing a wild-type mouse's macrophages with ApoE-deficientmacrophages accelerates atherosclerotic changes, even though the animalcontinues to produce ApoE by the liver. Fazio et al., Proc. Natl. Acad.Sci. 94:4647 (1997). In atherosclerosis it is hypothesized that ApoE,via a receptor-mediated event, downregulates macrophage activation inthe vicinity of blood vessel walls. Such down-regulation of macrophageactivation interrupts or interfers with the cascade of events associatedwith atherosclerotic plaque formation, to thereby reduce or slow theformation of atherosclerotic lesions. The cascade of events known to beassociated with atherosclerosis includes smooth muscle cell andendothelial cell proliferation, and foam cell formation; evidence existsthat ApoE downregulates each of these processes. ApoE thus affects thepresence and progression of atherosclerosis in vivo, independent of itseffects on lipids. The progression of atherosclerosis may be assessed bymeasuring the amount or size of atherosclerotic plaques, or thepercentage of the blood vessel blocked by an atherosclerotic lesion, orthe rate of growth of such plaques.

The present inventors have for the first time demonstrated that ApoEtransduces a calcium-mediated signal (Ca²⁺ /inositol triphosphate signaltransduction) in macrophage, indicating that ApoE modifies macrophagefunction by downregulating macrophage activation and, therefore,subsequent inflammation. Peptides, compounds, methods and pharmaceuticalformulations as described herein in relation to microglia and CNSdisease are accordingly useful in methods of suppressing the activationof macrophages to suppress, prevent, or slow atherosclerosis.Atherosclerosis refers to the thickening of the arterial intima andaccumulation of lipid in artherosclerotic plaques. Administration ofcompounds of the present invention to treat or prevent atherosclerosismay be by any means discussed herein as well as other suitable methodsthat are known in the art. When using the present compounds to prevent,slow or treat atherosclerotic changes, it is apparent that they need notbe formulated to pass through the blood brain barrier. Conditions thatmay be treated by the present method include atherosclerosis of thecoronary arteries; arteries supplying the Central Nervous system, suchas carotid arteries; arteries of the peripheral circulation or thesplanchnic circulation; and renal artery disease. Administration, suchas parenteral administration, may be site-specific or into the generalblood stream.

The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLE 1 Microglial Nitric Oxide Production Materials and Methods

This study examined the role of endogenous apoE in modulating microglialnitric oxide (NO) production, as measured by nitrite accumulationfollowing lipopolysaccharide (LPS) stimulation of microglia.

Culture preparation and characterization:

Mixed glial cell cultures were prepared from: (a) wildtype (C57/B16;Jackson Laboratories) mouse pups; (b) ApoE deficient mutant mouse pups(ApoE-deficient mice), and (c) transgenic mouse pups expressing humanApoE3 but not murine ApoE (ApoE3 mice). See Xu et al., Neurobiol. Dis.3:229 (1996) regarding the creation and characterization of thetransgenic mice. Mixed glial cell cultures were prepared as has beendescribed. See McMillian et al., Neurochem. 58:1308 (1992); Laskowitz etal., J. Neuroimmunol. 76:70 (1997). Briefly, brains were removed from2-4 day old pups, cleaned of membranes and blood vessels, mechanicallydispersed in Ca⁺²-free media, and collected by centrifugation. Cellswere then plated in DMEM/F12 (containing 10% fetal calf serum, 1%penicillin/streptomycin, Gibco #15070), one brain per 25 cm flask. Mixedneuronal/glial preparations were grown in humidified incubators untilconfluent (3-5 weeks).

The percentage of microglia, astrocytes and neurons were quantified todemonstrate that cultures prepared from ApoE-deficient and ApoE3 micehad comparable glial populations. Immunostaining was performed usingantibodies to glial fibrillary acidic protein (GFAP; SIGMA®; 1:500dilution) and tau protein (SIGMA®; 1:500 dilution) to estimate numbersof astrocytes and neurons, and peroxidase-coupled Bandeiraeasimplifolica B4 isolectin and naphthyl acetate esterase staining wasused to detect microglia. Laskowitz et al., J. Neuroimmunol. 76:70(1997). A mixed neuronal-glial culture system was used, as this mostclosely approximates the normal CNS milieu, and allows glia-gliainteractions, which play a role in the inflammatory cascade.

Comparable glial populations were confirmed using semi-quantitativeWestern blot analysis performed for astrocytes (αGFAP; SIGMA®), neurons((αtau; SIGMA®) and microglia (Bandeiraea simplifolica B4 isolectin;SIGMA®). Cellular protein was harvested at the end of experiments and 50μg protein from each sample was separated by polyacrilamide gelelectrophoresis and the protein was transferred to nylon membranes.Non-specific binding of antisera and lectin was blocked by preincubationof the membrane in 4% dried milk, 0.1% Triton X-100. Membranes wereincubated overnight with antibodies or 1 μg/ml B4 isolectin. Afterextensive washing in phosphate-buffered saline, bound antibody or lectinwas visualized by an ABC kit (Vector, Burlingame, CA), usingdiaminobenzidine as substrate.

Culture Stimulation:

Cultures were plated in serum-free media after washing cells once withthis media, and stimulated with LPS 100 ng/ml (SIGMA®). Aliquots weretaken at 24 and 60 hours for nitrite assay.

Nitrite Quantification:

The production of NO was assessed by measuring the accumulation ofnitrite, which was quantified using a colorimetric reaction with Griessreagent (0.1% N-1-naphthylethylenediamine dihydrochloride, 1%sulfanilamide, and 2.5% H₃PO₄). Absorbance was measured at 570 nm byspectrophotometry. The sensitivity of this assay is approximately 0.5μm.

Statistical Analysis:

Data were compared by ANOVA and the Fischer LSD multiple range test;p<0.05 was considered significant.

EXAMPLE 2 Microglial Nitric Oxide Production: Results

Culture Characterization:

No significant differences were found in glial populations among thecultures prepared from ApoE-deficient, ApoE3, and wild-type mice.Cultures comprised approximately 70% astrocytes, 15% microglia and 15%neurons. Comparisons of cellular preparations from wildtype mice,ApoE-deficient mice and ApoE3 mice showed no differences in glialpopulations. In particular, levels of microglia (the primary effectorcells for NO production) were comparable in all three culturepreparations, as detected by lectin binding (data not shown).

ApoE-deficient mouse cultures showed robust nitrite responses during thefirst 24 hours of exposure to LPS. This enhanced response was 6-foldgreater than that observed with microglia from control animals(p=0.0001; FIG. 1). Cultures from transgenic mice in which murine apoEis replaced with human ApoE3 show weak responses to LPS that were notsignificantly different than responses of wildtype animals (p=0.64 andp=0.2 at 24 and 60 hours, respectively). By 60 hours, increased nitriteaccumulation was observed in response to LPS in wildtype and ApoE3transgenic mouse preparations, although there was still a significantlygreater amount of nitrite in the apoE deficient culture as compared tocontrols (p=0.04%; FIG. 1)

The above studies show that ApoE deficient mixed neuronal-glial culturesrespond differently to LPS stimulation than glial cultures prepared frommice expressing native murine ApoE3 or those expressing the human ApoE3isoform. These results are consistent with ApoE being a biologicallyrelevant mediator of the CNS response to injury. These studiesdemonstrate that endogenous ApoE modulates glial secretion ofLPS-stimulated nitric oxide production, and suggest that one function ofendogenous ApoE produced within the brain is to suppress microglialreactivity and thus alter the CNS response to acute and chronic injury.

EXAMPLE 3 Suppression of Microglial Activation by Peptides of SEQ IDNO:3

Enriched microglia primary cultures were prepared from the brains ofapoE deficient mouse pups as described in Example 1, above. Themicroglia were stimulated with lipopolysaccharide (100 ng/ml) toactivate the microglia as described in Example 1. Activated microgliasecrete inflammatory cytokines and nitric oxide; the secretion of nitricoxide was used in the present experiment as a marker of microglialactivation. Nitric oxide production was assessed as described in Example1.

Peptides of SEQ ID NO:3 were added to cultures of activated microglia,in dosages of from 0 μM to 1000 pM. A dose-dependent decrease in nitricoxide secretion was observed after 48 hours (FIG. 2). The administrationof a peptide of SEQ ID NO:2 in a dose of 2mM did not result in anyapparent decrease in nitric oxide secretion (FIG. 2). The monomerpeptide of SEQ ID NO:2 acted as a control to establish that the observedresults are not due to any non-specific peptide effect.

EXAMPLE 4 Effect of ApoE an Macrophage

Intracellular signaling pathways of ApoE were investigated usingperitoneal macrophage.

Thioglycolate-elicited peritoneal macrophage were harvested from 8-weekold C57-BL6 mice, and plated at a density of 4×10⁵ cells on glasscoverslips, loaded with 2.5 μM Fura-2/AM for thirty minutes, and washedwith Hanks buffered solution containing 75 μM calcium. After exposure to5nM human recombinant apoE3 or E4, intracellular calcium was measured byZeiss digital microscopy. As shown in FIG. 3A, ApoE caused intracellularmobilization of intracellular calcium in the macrophage. Preincubationwith 100 molar excess of Receptor Associated Protein (RAP) did not blockthis effect; RAP is a physiological antagonist to LRP and blocks thefunction of LRP.

Macrophage were also plated at a density of 2×10⁶ cells/well, labeledwith ³H-myoinositol (8μC/ml) 16 hours at 37 degrees, and exposed tohuman ApoE3 or ApoE4 (5 nM). Control cells were exposed to vehicle butnot ApoE. Results are shown in FIG. 3B; values are expressed as thepercent change in inositol trisphosphate in treated cells as compared tocontrol cells.

Exposure of peritoneal macrophage to ApoE induced a rise inintracellular calcium associated with turnover of inositoltris-phosphate (FIGS. 3A and 3B). The present results indicate that ApoEinitiates a signal transduction pathway that affects and modifiesmacrophage function. The present data suggest that ApoE downregulatesmacrophage activation and inflammation; macrophage activation andinflammation is known to contribute to the atherosclerotic process.

EXAMPLE 5 Suppression of Microglial Activation Using Peptides of SEQ IDNO:6

A 20-amino acid peptide derived from the receptor binding region ofapoE, containing amino acids 130-149 (SEQ ID NO:6) was preparedaccording to methods known in the art.

Primary murine microglial cultures were prepared as described in Example1, from apoE deficient mouse pups. In some cultures the microglia wereactivated with lipopolysaccharide (100 ng/ml), as described in Example1.

Peptides of SEQ ID NO:6 were added to cultures of activated andnon-activated microglia, in dosages of 0 μM (control), 10 μM, 100 μM and1000 μM (FIG. 4). Each dosage level of peptide was tested alone(squares) and in combination with LPS (100 ng/ml; circles). Theproduction of TNFα was then measured 24 hours after addition of thepeptides. A decrease in TNFα production by activated microglia (comparedto control culture) was observed with each peptide dose used (FIG. 4,circles). Data in FIG. 4 is presented in at least triplicate at eachdose; error bars represent standard error of the mean).

These results indicate that peptides of SEQ ID NO:6 suppress cytokinerelease from activated glial cells.

EXAMPLE 6 Cytotoxicity of Peptides of SEQ ID NO:6

The toxic effects of peptides of SEQ ID NO:6 was investigated. Culturesof activated (LPS) and non-activated microglia, as described in Example5, were used. Peptides having SEQ ID NO:6 were added to cell cultures inamounts of 0 μM (control), 10 μM, 100 μM and 1000 μM; each dosage levelof peptide was tested alone (squares) and in combination with LPS (100ng/ml; circles). Cell viability was then measured by optical density 24hours after addition of the peptides.

As shown in FIG. 5, optical density was approximately the same incultures receiving 0 μM and 10 μM of peptide, but decreased in culturesreceiving 100 μM or 1000 μM. These results, taken with the results ofExample 5, indicate that a non-toxic concentration of a peptide of SEQID NO:6 is sufficient to suppress glial cytokine release.

EXAMPLE 7 Suppression of Glial Cytokines and Cytotoxicity of Peptides ofSEQ ID NO: 6

The experiments as described in Examples 5 and 6 were repeated using apeptide doses of 0 μM (control), 1 μM, 10 μM, 100 μM and 1000 μM. Eachdosage level of peptide was tested alone (squares) and in combinationwith LPS (100 ng/ml; circles). The production of TNFα was measured 24hours after administration of the peptides, and results are shown inFIG. 6. The optical density of the cell cultures was also measured (at24 hours) to assess cell viability; results are shown in FIG. 7.

These results show that microglial cytokine release was suppressed incell cultures receiving as little as 1 μM of peptide, but cytotoxiceffects were seen only in cultures receiving much larger doses ofpeptide. The results of examples 5-7 indicate that non-toxicconcentrations of peptides comprising the receptor binding region ofapoE are able to suppress cytokine release from activated microglia.

EXAMPLE 8 In vivo Treatment of Focal Ischemia

A murine model of focal ischemia-reperfusion is used to assess theeffects of intrathecal, intravenous or intraperitoneal administration ofsmall therapeutic peptides (fewer than 30 amino acids in length)comprising the apoE LDL receptor region. One such peptide has SEQ IDNO:6.

Wild-type mice are subjected to middle cerebral artery occlusion andreperfusion according to techniques known in the art (see, e.g,Laskowitz et al., J. Cereb. Blood Flow Metab. 17:753 (July 1997)). Onegroup of mice (wild-type control) receives no treatment after cerebralartery occlusion; in a similar group (wild-type treatment group) eachmouse receives intrathecal, intraperitoneal or intravenous injection ofa therapeutic peptide. Therapeutic peptides may be injected in varyingdoses, using the in vitro data provided above as an initial guide.

Each animal is evaluated neurologically at a predetermined time afterreperfusion (e.g., 24 hours after reperfusion) (see, e.g. Laskowitz etal., J. Cereb. Blood Flow Metab. 17:753 (July 1997)). After neurologicalexamination each mouse is anesthetized and sacrificed and the brain issectioned and stained, and infarct volume is measured. Neurologicaloutcome and infarct size is compared between control and treatmentgroups.

The above experiments may be repeated using apoE deficient mice.

EXAMPLE 9 In vivo Treatment of Global Ischemia

A murine model of global ischemia, adapted from the rat two vesselocclusion model of global ischemia, is used to assess the effects ofintrathecal administration of small therapeutic peptides (fewer than 30amino acids in length) comprising the apoE LDL receptor region. One suchpeptide has SEQ ID NO:6.

Wild-type mice (21±1 grams) are fasted overnight, anesthetized withhalothane or another suitable anesthetic, intubated and mechanicallyventilated. The right internal jugular vein and femoral artery arecannulated. Pericranial temperature is held at 37.0° C. The carotidarteries are occluded and mean arterial pressue is reduced to 35 mmHgwith 0.3 mg intra-arterial trimethaphan and venous exsanguination. Tenminutes later ischemia is reversed. Control mice receive no additionaltreatment, test mice receive intrathecal, intravenous or intraperitonealinjection of a therapeutic peptide. Peptides may be injected at varyingdoses, using the in vitro data provided herein as a guide.

Each animal is evaluated neurologically at a predetermined time (e.g.,1, 3 or 5 days after reperfusion), using known neurological testingprocedures (see, e.g., Laskowitz et al., J. Cereb. Blood Flow Metab.17:753 (July 1997)). After neurological evaluation, each animal isanesthetized and sacrificed and the brain injury is assessed usingmethods known in the art. For example, brains may be perfusion fixed insitu, then sectioned, stained and examined by light microscopy, forexample, to determine injury to the CA1 sector of the hippocampus, andviable and non-viable neurons counted and compared.

Neurological outcome and brain injury is compared between control andtreatment groups.

EXAMPLE 10 Apolipoprotein E and apoE-Mimetic Peptides Initiate aCalcium-Dependent Signaling Response in Macrophages

This example shows that apoE initiates a signaling cascade in murineperitoneal macrophage that is associated with mobilization ofintracellular Ca²⁺ stores following increased production of inositoltrisphosphate. This cascade was inhibited by pretreatment withreceptor-associated protein and Ni²⁺. Signal transduction was mediatedby a pertussis toxin-sensitive G protein. These are characteristicproperties of signal transduction induced via ligand binding to thelipoprotein receptor-related protein (LRP) receptor. A peptide derivedfrom the receptor binding region of apoE also initiated signaltransduction in the same manner as the intact protein. The presence ofcross desensitization suggested that the apoE and the apoE-mimeticpeptide competed for the same binding site. This was confirmed by ourobservation that radiolabeled apoE-mimetic peptide competed with theintact protein for receptor binding. These data indicates thatApoE-dependent signal transduction mediates the immunomodulatoryproperties of this lipoprotein.

A. Materials And Methods

Materials.

Brewer's thioglycollate broth was purchased from Difco Laboratories(Baltimore, MD). RPMI Medium 1640, fetal bovine serum, Hanks' BalancedSalt Solution and other cell culture reagents were purchased from LifeTechnologies, Inc. (Grand Island, NY). Bovine serum albumin (BSA),pertussis toxin, and HEPES were from Sigma Chemical Co. (St. Louis,Mo.). Fura-2AM and BAPTA/AM were obtained from Molecular Probes (Eugene,Oreg.). Myo-[2-³H]inositol (specific activity 10-20 Ci/mmol) waspurchased from American Radiolabeled Biochemicals (St. Louis. Mo.). Aplasmid containing the RAP cDNA was a kind gift from Dr. Joachim Herz,the University of Texas, Southwestern, Dallas Tex. It was used toproduce RAP as previously described [21]. Human recombinant apoE2 wasobtained commercially from Panvera Corp (Madison, Wis.). The preparationwas free of endotoxin, and homogenous as judged by SDS-polyacrylamidegel electrophoresis. [³H]thymidine (specific activity, 70 Ci/mmol) andIodine-125 (specific activity: 440 mCi/mg) were purchased from theAmerican Radiolabeled Chemicals, Inc. (St. Louis MO). The 20 amino acidApoE mimetic peptide (Ac-TEELRVRLASHLRKLRKRLL-amide) with and without atyrosine on the amino terminus as well as a scrambled control peptide ofidentical size, amino acid composition, and purity were synthesized byQCB Biochemicals (Hopkinton, Mass.) to a purity of 95%. All aminotermini were acetylated and all carboxyl termini were blocked with anamide moiety. Peptides were reconstituted in sterile isotonic phosphatebuffered saline. A scrambled control peptide of identical size, aminoacid composition, and purity was also synthesized. All other reagentsused were of the highest quality commercially available.

Macrophage Harvesting.

All experiments involving animals were first approved by the DukeInstitutional Animal Care and Use Committee. Pathogen-free femaleC57BL/6 mice and ApoE deficient mice previously backcrossed 10 times tothe C57BL/6 strain were obtained from the Jackson Laboratory (BarHarbor, Maine). Thioglycollate-elicited peritoneal macrophages wereharvested by peritoneal lavage using 10 ml of ice-cold Hanks' balancedsalt solution containing 10 mM HEPES and 3.5 mM NaHCO₃ (HHBSS), pH 7.4.The macrophages were pelleted by centrifugation at 4° C. at ˜800×g for10 min and resuspended in RPMI 1640 media supplemented with 25 mM HEPES,12.5 U/ml penicillin, 6.5 mg/ml streptomycin, and 5% fetal bovine serum.Cell viability was determined by the trypan blue exclusion method andwas consistently greater then 95%.

Receptor Binding Studies.

Macrophages were plated in 48-well cell culture plates (Costar) at2.5×10⁵ cells per well and incubated for 3 h at 37° C. in a humidified5% CO₂ incubator. The plates were then cooled to 4° C. and unbound cellswere removed by three consecutive rinses with ice-cold Hanks' balancedsalt solution containing 20 mM Hepes and 5% BSA, pH 7.4 (bindingbuffer). To quantify direct binding of the ¹²⁵I-apoE mimetic peptide,varying amounts of radiolabeled peptide were added to each well in thepresence or absence of 200-fold molar excess of unlabeled peptide.Specific binding to cells was determined by subtracting the amount of¹²⁵I-apoE peptide bound in the presence of excess unlabeled peptide(nonspecific binding) from the amount of ¹²⁵I-apoE peptide bound in theabsence of excess unlabeled peptide (total binding). For competitionstudies, 50 nM radiolabeled peptide was added to each well in thepresence or absence of varying amounts (31.25 nM -4 □M) of unlabeledApoE2 or RAP. Cells were then incubated at 40° C. for 12-16 h. Unboundligand was removed from the wells and the cell monolayer was rinsedthree times with ice-cold binding buffer. Cells were then solubilizedwith 1 M NaOH, 0.5% SDS at room temperature for >5 h before the contentsof each well was added to polystyrene tubes and counted in a LKB-Wallac,CliniGamma 1272 □-counter (Finland).

Measurement of [Ca²⁺]_(i) in apoE and peptide treated macrophage.

Changes in [Ca²⁺]_(i) levels in Fura-2/AM treated single cells werequantified using digital imaging microscopy in accordance with knowntechniques. Macrophages were plated on glass coverslips sitting in 35 mmPetri dishes at a density of 1.5×10⁵ cells/cm², and allowed to adherefor 2 h in a humidified 5% CO₂ incubator at 37° C. The non-adherentcells were aspirated and the monolayers were washed twice with HHBSS. 4μM Fura-2/AM was incubated with the cells for thirty min in the dark atroom temperature and [Ca²⁺]_(i) was subsequently measured using adigital imaging microscope in accordance with known techniques. Afterobtaining baseline measurements for 5 min, ligand (apoE, apoE mimeticpeptide, or scrambled peptide) was added, and multiple [Ca²⁺]_(i)measurements were taken. To determine if signaling resulted fromligation of the ligand to LRP, cells were preincubated with a 1000-foldmolar excess of RAP or 10 mM NiCl₂, both of which inhibit ligand bindingto LRP, for 5 min prior to stimulation with apoE or peptide. Inexperiments in which the involvement of a G protein was assessed,monolayers were incubated with 1 μg/ml pertussis toxin for 12 h at 37°C. and Ca²⁺ measurements were made as stated above.

Measurement of IP₃ in apoE treated macrophage and effect of pertussistoxin.

The formation of IP₃ in myo-[2-³H]inositol-labeled macrophages undervarious experimental conditions was quantified in accordance with knowntechniques. Macrophage were plated in 6 well plates (4×10⁶ cells/well)and allowed to adhere at 37° C. for 2 h in a humidified 5% CO₂incubator. Medium was aspirated from the monolayers and RPMI 1640 mediumcontaining 0.25% BSA and myo-[2-³H]inositol (specific activity 10-20Ci/mmol) was added to each well. The cells were incubated at 37° (C. foran additional 16-18 h. Monolayers were rinsed three times with 25 mMHHBSS containing 1 mM CaCl₂, 1 mM MgCl₂, 10 mM LiCl, pH 7.4. A volume of0.5 ml of this solution was added to each well, and the cells werepreincubated for 3 min at 37 ° C. before stimulated with ligand. Thereaction was stopped by aspirating the medium containing the ligand andadding 6.25% perchloric acid. The cells were scraped out of the wells,transferred to tubes containing 1 ml of octylamine/Freon (1:1 vol/vol)and 5 mM EDTA, and were centrifuged at 5600×g for 20 min at 4° C. Theupper phase solution was applied to a 1ml Dowex resin column (AG1-X8formate; Bio Rad Laboratories, Richmond, CA) and eluted sequentially inbatch process with H₂O, 50, 200, 400, 800, and 1200 mM ammonium formatecontaining 0.1 M formic acid [26]. Radioactivity was determined byplacing aliquots in a liquid scintillation counter to determineradioactivity. To evaluate the pertussis-toxin sensitivity of the Gprotein coupled to receptor activation and phosphatidyl inositol4,5-bisphosphate (PIP2) hydrolysis, cells were plated as described aboveand incubated with 1 μg/ml pertussis toxin which had been preactivatedwith 40 mM DTT at 30° C. for 20 min. The effect on IP₃ formation wasmeasured as described above.

Competition between apoE and apoE mimetic peptide for binding site onthe receptor.

Changes in macrophage [Ca²⁺]_(i) upon stimulation with apoE andapoE-mimetic peptide were studied to determine whether these ligandsbind to the same receptor. Fura-2/AM loaded macrophages were incubatedovernight, plated on glass cover slips, stimulated with one ligand, andchanges in [Ca²⁺]_(i) quantified. Cells were then stimulated with secondligand and Ca²⁺ measurements repeated

B. Results

Effect of apoE on macrophage [Ca²⁺]_(i).

Modulation of free cytoplasmic Ca²⁺ concentration is a ubiquitoussignaling response. In many cell types, binding of ligands to plasmamembrane receptors activates the hydrolysis of PIP₂ by membrane-boundphospholipase C, generating IP₃. IP₃ causes the release of Ca²⁺ from theendoplasmic reticulum by binding to its cognate receptor, which is alsoa Ca²⁺ channel. In non-excitable cells, [Ca²⁺]_(i) signaling isassociated both with Ca²⁺ release from intracellular stores and Cainflux. Treatment of macrophages with human recombinant apoE increased[Ca²⁺]_(i) levels 2-4-fold compared to macrophage treated with buffer(FIG. 8A). In a typical experiment [Ca²⁺]_(i) levels in unstimulatedcells and apoE-treated cells were 95.33±7.37 and 180.25±14.57 nM,respectively. The increase in [Ca²⁺]_(i) upon stimulation with apoE wasobserved in 70-80% of the cells examined. ApoE-induced increase in[Ca²⁺]_(i) was heterogeneous, asynchronous, and either oscillatory orsustained. ApoE-induced increases in macrophage [Ca²⁺]_(i) wasdose-dependent (FIG. 8B). To address the possibility that native apoEsecreted by macrophage altered responses to exogenous human recombinantapoE, these experiments were repeated using macrophage prepared fromapoE deficient mice. Calcium responses following stimulation with apoEwere identical in wild-type macrophages and macrophages from apoEdeficient mice (data not shown).

The effect of pertussis toxin on apoE-induced IP₃ synthesis.

Exposure of myo-[2-³H] inositol-labeled macrophage to apoE caused a1.5-2.0-fold increase in IP₃ levels (FIG. 9A). This effect wasdose-dependent (FIG. 9B). Pretreatment of the macrophages with pertussistoxin completely abolished this increase in IP₃. (FIG. 9A). Thesestudies demonstrate that the phospholipase C-catalyzed hydrolysis ofmembrane PIP₂ in apoE stimulated cells is coupled to a pertussistoxin-sensitive G protein.

ApoE-induced increases in macrophage [Ca²⁺]_(i) are attenuated by Ni²⁺and RAP.

Previous studies have demonstrated that ApoE binds to LRP and is theninternalized. Additionally, binding of lactoferrin, Pseudomonas exotoxinA, lipoprotein lipase and thrombospondin to LRP initiates a signalingcascade associated with the generation of second messengers. Toinvestigate the possibility that LRP is involved in the signal cascadeinduced by apoE, macrophages were preincubated with RAP and Ni⁺²prior tostimulation with apoE2 or apoE2 mimetic peptide. RAP is a 39 kD proteinthat blocks the binding of all known ligands to LRP. Ni²⁺ also blocksligand interactions with LRP. Both preincubation with RAP and Ni⁺²markedly attenuated the [Ca²⁺]_(i) increases associated with subsequentexposure to apoE (data not shown). These results are consistent with thehypothesis that apoE induces a signaling cascade via specificinteraction with LRP. Pretreatment of macrophage with pertussis toxinalso markedly attenuated the ApoE-dependent Ca²⁺ response, indicatingthat signal transduction induced by apoE is coupled to a pertussistoxin-sensitive G protein. This is consistent with the known propertiesof LRP-dependent signal transduction.

Effect of apoE-mimetic peptide on macrophage [Ca²⁺]_(i).

Stimulation of macrophage with the peptide derived from residues 130-149of the apoE receptor binding region also resulted in a 2-3-fold increasein [Ca²⁺]_(i) whereas a scrambled control peptide of identical size andcomposition had no effect (data not shown). This increase in [Ca²⁺]_(i)was observed in approximately 60-70% of cells examined. As with the apoEresponses, peptide-induced increases in macrophage [Ca²⁺]_(i) wereheterogeneous and asynchronous. These results demonstrate that bothintact apoE and a peptide derive from the apoE receptor binding regioninduce an increase in [Ca²⁺]_(i) that is consistent with the initiationof a signaling cascade. However, on a molar basis, higher concentrationsof peptide were necessary to get [Ca²⁺]_(i) responses compared to theintact apoE. This difference likely results from differences in receptoraffinity between the peptide and apoE, a property generally seen whencomparing the effects of intact proteins to peptide ligands.

Effects of repeated stimulation of apoE and apoE-mimetic peptide on[Ca²⁺]_(i).

We evaluated the possibility of competition between apoE and its mimeticpeptide for binding sites on the receptor by quantifying the changes in[Ca²⁺]_(i) consequent to receptor ligation. Following repeated exposureto apoE, there was a marked attenuation in [Ca²⁺]_(i) suggestingtachyphylaxis (data not shown). Following the increase in [Ca²⁺]_(i)associated with the initial exposure to human recombinant apoE, therewas a marked attenuation in [Ca²⁺]_(i) response to subsequent peptideexposure (data not shown). Similarly, there was a loss of [Ca²⁺]_(i)response to apoE addition following initial exposure to peptide (datanot shown). No desensitization in calcium response was observed withexposure of scrambled peptide (data not shown). This observedtachyphylaxis suggests receptor desensitization secondary to receptorligation, and is consistent with the hypothesis that both the intactapoE protein and the 20 residue peptide bind to the same receptor.

C. Discussion

The primary observations of this example are that: 1) binding toreceptors on the macrophage cell surface of human recombinant apoE (inpM to nM concentrations) initiates signaling events associated withincreases in [Ca²⁺]_(i) and IP₃; 2) a 20 residue peptide derived fromthe receptor binding region of apoE, but not a scrambled controlpeptide, causes identical changes in macrophage [Ca²⁺]_(i); 3) changesin [Ca²⁺]_(i) and IP₃ are specific and dose-dependent; 4) apoE-inducedincrease in cellular IP₃ is pertussis toxin-sensitive; and 5) changes in[Ca²⁺]_(i) are blocked by RAP and Ni²⁺. Moreover, based on the presenceof cross-desensitization, apoE and the apoE-mimetic peptide appear tobind to the same receptor.

EXAMPLE 11 An Apolipoprotein E Mimetic Peptide is Protective in a MurineHead Injury Model

This Example demonstrates a protective effect of intravenousadministration of a 17 amino acid apoE mimetic peptide (the fragment ofApoE containing amino acids 133-149) following head injury.

Mice were endotracheally intubated and their lungs were mechanicallyventilated with 1.6% isoflurane at 30% partial pressure of oxygen. Themice received a midline closed head injury delivered by a pneumaticimpactor at a speed of 6.8 m/s. Thirty minutes after closed head injury,mice were randomized into 3 groups (n=16 mice per group as follows: highdose peptide (406 ug/kg), low dose peptide (203 ug/kg), and salinecontrol solution. All peptide solutions were prepared in sterileisotonic saline (100 ul) and delivered intravenously via tail veininjection. Rotorod time and weight were measured for five consecutivedays after injury. At 21 days, the ability to learn to find a hiddenplatform in the Morris Water Maze was tested.

Prior to injury, rotorod latency and weights were comparable in allanimals. After injury, the saline injected animals had a profounddeficit in rotorod testing which was associated with weight loss. Highdose peptide, and to a lesser extent low dose peptide protected animalsfrom this motor deficit (FIG. 10A), and concomitant weight loss (FIG. 10b). This protective effect of the single dose of peptide was sustainedfor five days following injury (p<0.05 3-way repeat measures ANOVA).

In addition, the peptide appeared to provide protection in learningdeficits in learning to find a hidden platform (FIG. 10C) in the MorrisWater Maze (p<0.05 3-way repeat measures ANOVA). Treatment with thepeptide also resulted in a significant improvement in acute survival asdemonstrated by Kaplan-Meier analysis (FIG. 10D).

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof.. The invention is describedby the following claims, with equivalents of the claims to be includedtherein.

1-13. (canceled)
 14. A therapeutic peptide containing the sequence ofSEQ ID No. 10, wherein said peptide suppresses microglial activation.15. The peptide of claim 14, wherein said peptide contains SEQ ID No. 10linked to one to five additional amino acids or amino acid analogues atthe N-terminus or C-terminus or both the N- and C-terminus, wherein suchadditional amino acids do not adversely affect the therapeutic functionof the peptide.
 16. The peptide of claim 14, wherein said peptidecontains 18 amino acids or more.
 17. The peptide of claim 14, whereinsaid peptide contains 20 amino acids or more.
 18. The peptide of claim14, wherein said peptide contains 30 amino acids or more.
 19. Thepeptide of claim 14, wherein said peptide contains 40 amino acids ormore.
 20. The peptide of claim 14, wherein said peptide consistsessentially of SEQ ID No.
 10. 21. A composition comprising the peptideof claim 14 in combination with a pharmaceutically acceptable carrier,diluent or excipient.
 22. The composition of claim 21, wherein saidpeptide is conjugated to a carrier molecule that increases transport ofsaid compound across the blood-brain barrier, compared to that whichwould occur in the absence of said carrier molecule.
 23. The compositionof claim 21, wherein said carrier is selected from the group consistingof pyridinium, fatty acids, inositol, cholesterol, glucose derivatives,hemoglobin, lysozyme, cytochrome c, ceruloplasmin calmodulin ubiquitin,substance P, histone, insulin and transferrin and peptides thereof,lipid vesicles and liposomes.